MicroRNAs (also referred to herein as “miRNA”s) function in gene regulation by regulating mRNA transcript stability and mRNA translation to proteins. A miRNA is complementary to a part of one or more messenger RNAs (mRNAs). Animal miRNAs are usually complementary to a site in the 3′ UTR where the microRNAs partially base pair and inhibit protein translation of the target mRNA. MicroRNAs that are partially complementary to the target can also speed up deadenylation, causing mRNAs to be degraded sooner. For partially complementary microRNA to recognise their targets, the nucleotides 2-7 of the miRNA (‘seed region’), generally have to be highly complementary. MiRNAs occasionally also causes DNA methylation of promoter sites and therefore affecting the expression of targeted genes. MiRNAs function in association with a complement of proteins collectively termed the miRNP. Human miRNPs contain eIF2C2 (also known as Argonaute 2), DDX20, GEMIN4 and microRNA.
Animal microRNAs target particular developmental genes. In contrast, genes involved in functions common to all cells, such as gene expression, have very few microRNA target sites, and seem to be under selection to avoid targeting by microRNAs.
As of 2002, miRNAs had been confirmed in various plants and animals, including C. elegans, human and the plant Arabidopsis thaliana. There has been production of microarrays (e.g., dubbed MMChips) containing all the known miRNAs for human, mouse, rat, dog, C. elegans and Drosophila. Agilent and other firms have subsequently commercialized human miRNA microarrays.
MicroRNA expression can be quantified by modified RT followed by qPCR, or profiled against a database describing thousands of known miRNAs using microarray technology. The activity of a miRNA can be experimentally inhibited using a locked nucleic acid oligo, a Morpholino oligo or a 2′-O-methyl RNA oligo. MicroRNA maturation can be inhibited at several points by steric-blocking oligos. The miRNA target site of an mRNA transcript can also be blocked by a steric-blocking oligo. Additionally, a specific miRNA can be silenced by a complementary antagomir.
MicroRNAs regulate mRNA transcript stability and mRNA translation to proteins. The binding of a specific microRNA to a given target mRNA is difficult to directly affirm using currently available techniques. Using conventional informatics approaches, publically available sequence software is notoriously unreliable, exemplified in that each program differs dramatically in prediction of binding of individual microRNAs to a given mRNA transcript. This is in part due to the tendency of short microRNA oligos to have high homology to many transcriptome and genomic sequences as well as the subtle sequence differences among microRNAs of one or very few bases. Experimentally, microRNAs can bind cDNA oligomers on a spotted microarray, or alternately a particular cDNA, with a number of mismatches; these cDNA-based methods are both non-specific, and do not measure the interaction of biological interest (microRNA:mRNA binding). The binding of a specific microRNA to a given target mRNA is at the crux of transcript stability and translational regulation. Identification of microRNAs which bind to a given target mRNA will allow the use of microRNAs for targeted repression of mRNA translation and degradation of mRNAs, thereby affecting gene expression. Specific microRNA-mRNA binding is very difficult to affirm using currently available techniques. Informatics approaches are of variable reliability. Experimental approaches are largely non-existent. The present invention addresses this need.